Enteral liquid ventilation oxygenates a hypoxic pig model

Summary The potential of extrapulmonary ventilation pathways remains largely unexplored. Here, we assessed the enteral ventilation approach in hypoxic porcine models under controlled mechanical ventilation. 20 mL/kg of oxygenated perfluorodecalin (O2-PFD) was intra-anally delivered by a rectal tube. We simultaneously monitored arterial and pulmonary arterial blood gases every 2 min up to 30 min to determine the gut-mediated systemic and venous oxygenation kinetics. Intrarectal O2-PFD administration significantly increased the partial pressure of oxygen in arterial blood from 54.5 ± 6.4 to 61.1 ± 6.2 mmHg (mean ± SD) and reduced the partial pressure of carbon dioxide from 38.0 ± 5.6 to 34.4 ± 5.9 mmHg. Early oxygen transfer dynamics inversely correlate with baseline oxygenation status. SvO2 dynamic monitoring data indicated that oxygenation likely originated from the venous outflow of the broad segment of large intestine including the inferior mesenteric vein route. Enteral ventilation pathway offers an effective means for systemic oxygenation, thus warranting further clinical development.


INTRODUCTION
For progressive respiratory failure, oxygen therapy can save the patient's life. However, approximately onequarter of hospitals surveyed in resource-limited countries lack sufficient oxygen supply. 1 This limitation is becoming more obvious with recent COVID-19 pandemic as patients regularly require interhospital transport for diagnostic or therapeutic purposes accelerated by hospital overcrowding. Although there are transient oxygen delivery methods available, several major limitations are important to take into account. For example, patients are at risk for severe respiratory distress or exhaustion due to increased breathing load. 2 Therefore, a simple and less invasive lung-independent respiratory support is highly demanding particularly in areas with resource-poor settings.
Historically, extrapulmonary oxygenation routes involving the gastrointestinal (GI) tract have been clinically explored. In the 1950s, for example, gastric gas insufflation was tested to treat asphyxia in newborns. [3][4][5][6][7] However, the effectiveness of enteral ventilation therapy remains widely debated. 8 Several groups recently revisited the potential of the enteral route for the systemic delivery of oxygen. Using a liquid solvent called perfluorocarbons with extraordinary oxygen solubility, Okabe et al. showed that intrarectal administration of oxygenated perfluorocarbons led to systemic oxygenation in animals with respiratory failure. 9 Another preprint study by Mountford et al. demonstrated that delivering oxygen microbubbles increased systemic blood oxygen levels in smoke-inhaled hypoxic pig models. 10 While the methods above seem promising, GI-mediated systemic oxygenation is compounded by the recipient's remaining lung function. Therefore, it is imperative to understand parameters that influence the gas exchange efficiency and exclude lung contribution to determine the gut-mediated oxygenation potential.
Herein, we evaluated the effectiveness of the enteral ventilation approach under controlled mechanical ventilation. To dissociate contributions from native lung function, we applied a muscle relaxant in a stable hypoxic minipig model. We investigated whether GI-mediated systemic oxygenation is delivered exclusively from the gut or whether the impact is combined with improved native lung function. Arterial pressure, pulmonary arterial pressure, oxygen saturation (SpO 2 ), heart rate, blood temperature, and mixed venous oxygen saturation (SvO 2 ) were continuously monitored. The objective of this study was to evaluate the extrapulmonary ventilation pathway in a large animal model to set the stage for future clinical development.

RESULTS
The experimental outlines are illustrated in Figure 1. The profiles of the nine pigs are summarized in Table 1. We used two differently sized pigs in our experiments to evaluate whether body weight affects the oxygenation effect. In addition, we conducted experiments to confirm the local oxygenation using normal-sized pigs due to the practicality of portal vein cannulation. To introduce hypoxia, FiO 2 was set at 0.11-0.15. At baseline, SaO 2 , PaO 2 , and PaCO 2 were 82.5G7.3%, 54.5G6.4mmHg, and 38.0G5.6 mmHg, respectively. Other parameters such as body temperature and blood pressure that might affect the oxygenation curve were carefully controlled throughout the procedures. We bubbled pure oxygen gas into the perfluorodecalin (PFD) liquid to load oxygen into PFD. The partial pressure of oxygen in PFD reached near saturation (760 mmHg) 5 min after the bubbling ( Figure 1A). Oxygen levels in PFD were retained at high levels (>650 mmHg) for one week in an administration bag ( Figure 1B). Intrarectal administration of oxygenated PFD (O 2 -PFD) was repeated three times in each pig, and 27 experiments were performed.

Correlative factors for oxygenation
Throughout the experiments, immediate oxygenation response was detectable after O 2 -PFD administration in minipigs. Therefore, we first evaluated the early oxygenation dynamics, noting that oxygen transfer peaked around the first 4 min. Mean 4 min-increase in SvO 2, SaO 2 , and PaO 2, from the baseline was +4.56%, +3.65%, and +3.60 mmHg, respectively. We next explored the correlation between (C) Schematic diagram of the experimental protocols. Rectal O 2 -PFD administration was repeated 3 times, accompanied by collection twice. Data are represented as mean G SD N 2 , nitrogen; O 2 , oxygen; O 2 -PFD, oxygenated perfluorodecalin; min, minutes; PO 2 , partial pressure of oxygen; SpO 2 , pulse oximetry; PaO 2 , partial pressure of oxygen in arterial blood; SaO 2 , arterial oxygen saturation; SvO 2 , mixed venous oxygen saturation; SivcO 2 , oxygen saturation of the inferior vena cava; SpvO 2 , oxygen saturation of the portal vein. iScience Article time-evolving oxygenation parameters (at 2, 4, 8, and 12 min after the administration) and the pre-treatment gas parameters to understand the variables for defining oxygen transfer efficiency. All the Pearson correlation coefficients are shown in Figure S1A. For example, the baseline PaO 2 level showed an inverse correlation with a 4 min-increase in arterial oxygenation (delta (D)PaO 2 4min) with a correlation coefficient of À0.475. When we limit the baseline PaO 2 level to 45À65 mmHg, its linearity improves to À0.740. Notably, of all the tested parameters, the baseline SvO 2 was most closely correlated with the transfer of oxygen, with a linear correlation coefficient of À0.728 with DSaO 2 4min of O 2 -PFD administration ( Figure S1B). These results indicate the enteral ventilation effect starts as early as 2-4 min, and its potency is inversely associated with prior oxygenation status, particularly at the level of the vein.

Peaked impacts on O 2 uptake and CO 2 removal
We next determined the time-course dynamics of each gas parameter every 2 min in minipigs. The enteral ventilation effect to improve oxygenation was sustained for up to 30 min until PFD was removed from the gut (Figure 2A), while respiratory rate and minute ventilation volume remained unchanged ( Figure 2B). The maximum effect on systemic oxygenation was 88.2 G 5.4% compared to the baseline of 82.5 G 7.3 (p < 0.001) for SaO 2 . PaO 2 also increased from 54.5 G 6.4 to 61.1 G 6.2 mmHg (p < 0.001), indicating the resolution of the induced respiratory failure conditions ( Figure 3A). Given that PFD dissolves significant amounts of most gases, including carbon dioxide, typically in the 120À250 vol % range, i.e., 3-5 times more than oxygen, we tested if enteral O 2 -PFD has effects on carbon dioxide removal. Interestingly, PaCO 2 was significantly reduced throughout the experiment: 38.0 G 5.6 at the baseline and 34.4 G 5.9 mmHg at post-O 2 -PFD administration (p < 0.001) ( Figure 3B). In particular, the increase in oxygenation showed a higher trend in mixed venous blood than in arterial blood throughout treatment (3.4 G 5.5% in DSaO 2 versus 4.9 G 6.3% in DSvO 2 , at 8 min after O 2 -PFD infusion, p = 0.082), suggesting that the oxygen uptake precedes the arterial oxygenation via venous route.
Such systemic ventilatory effects were rapidly reversed upon removal of the enteral PFD in 30 min. To assess the changes in the dissolved oxygen and carbon dioxide in PFD, we measured the partial pressure of the collected PFD. The oxygen partial pressure was significantly lower after 30 min of administration (from 688 G 36 to 395 G 96 mmHg after collection; p < 0.001), whereas the carbon dioxide partial pressure was increased (3.4 G 1.1 to 15.0 G 6.2 mmHg after collection; p < 0.001) ( Figures 3C and 3D). Conversely, SaO 2 decreased from 89.0 G 5.0% to 81.2 G 7.9% (p < 0.001), and PaO 2 decreased from 61.1 G 6.5 to iScience Article 53.6 G 7.0 mmHg (p < 0.001), while PaCO 2 increased from 34.8 G 5.9 to 37.9 G 6.7 mmHg (p < 0.001). The post-retrieval trend of rapid oxygen reduction was similar to the change at the time of O 2 -PFD administration ( Figure 2A). These results indicate that oxygen and carbon dioxide were exchanged by enteral exposure to PFD.

Venous oxygenation through the distal intestine
Inspiratory and exhaled oxygen concentrations were stabilized before the administration of O 2 -PFD. Respirator settings remained unchanged throughout the procedures ( Figure 2B) and inspiratory oxygen concentration was consistent (FiO 2 , from 12.9 G 1.7% to 13.0 G 1.7% (p = 0.164)). However, the exhaled oxygen concentration was increased from 8.1 G 1.3% (baseline) to 8.9 G 1.4% (maximum) (p < 0.001) following the administration of O 2 -PFD. The resultant difference between inspiratory and exhaled oxygen concentration reduced from 4.8 G 0.9% to 4.1 G 0.9% (p < 0.001). A significant increase in exhaled oxygen concentration indicates that enteral ventilation might oxygenate via returning, i.e., venous, circulatory route.
To further explore how enteral ventilation impacts venous oxygenation dynamics in blood at different anatomical levels, we performed the gas analysis of the pulmonary artery, IVC, and portal vein, before and after treatment in normal-sized pigs ( Figure 4A). Colorectal venous drainage systems are governed by the superior, middle, and inferior rectal (hemorrhoidal) veins. The superior rectal and inferior mesenteric veins drain into the portal vein, passing the blood through the liver before reaching the systemic circulation. In contrast, the inferior and middle rectal veins drain into the inferior vena cava and, therefore, directly into the systemic circulation, which is approximately responsible for À50% of suppository drug absorption. 11 Figure 4B). Interestingly, the venous oxygen saturation in both the portal vein and inferior vena cava showed upward trends until 14 min and then gradually declined while sustained higher level than the baseline ( Figure 4B), indicating local oxygen imbalance between supply and consumption occurred in

Adverse events
The gross examination showed no signs of colorectal damage or perforation after repeated dosing. Consistently, histopathological examination revealed no damage or inflammation to the rectal mucosa due to PFD (Figures S2A-SC). In addition, no macrophages that phagocytized PFD were observed in the spleen (Figures S2D and S2E).
During enteral ventilation under hypoxia, hemodynamics studies showed that mean arterial pressure increased and pulse rate decreased significantly. Maximal vital sign changes induced by O 2 -PFD administration ranged from 115 G 27 to 129 G 26 mmHg (p < 0.001) in the mean arterial pressure  Figure S3). It is not clear whether MAP increase effect is due to an oxygenating effect or an effect of an increased venous return. Thus, despite severe hypoxia, the animals did not undergo shock, and, in fact, the mean arterial pressure increased by an average of approximately 10 mmHg.

DISCUSSION
Our study, using hypoxic pig models with a combination of approved medical devices and a clinically feasible dose of O 2 -PFD, provides definitive proof that enteral ventilation is possible, independent of the effect of native lung respiration. Initial oxygen transfer and carbon dioxide reduction started within 2 min of O 2 -PFD administration and sustained blood gas status until PFD was removed at 30 min. SvO 2 dynamic monitoring data indicated that oxygenation most likely started from the venous outflow of the gut, as expected. We also found an increase in oxygen in the portal vein, suggesting potential uptake through the broader large intestine and the inferior mesenteric vein route, even above the level of the rectum. Therefore, extrapulmonary respiration induces systemic oxygenation through venous oxygenation.
Among aquatic organisms that utilize GI as extrapulmonary respiratory organs, the loach has a gas exchange function in its hindgut (distal intestine). 12,13 Such gut air-breathing (GAB) species use their gastrointestinal system to maintain systemic oxygenation under respiratory distressed conditions. 14 Though precise mechanisms for hypoxia acclimation are unclear, aerial ventilation in GAB fishes is driven primarily by oxygen partial pressure of the water (PO 2 ). 14 For example, the onset of aerial respiration occurred at inspired oxygen tensions (P i O 2 ) between 50 and 60 mmHg, and breathing frequency is linearly increased to 25 mmHg without significant hypometabolic change in Hypostomus regani. 15 Our pig study similarly suggested that the prior hypoxic state influences the mammalian ventilatory effect. Indeed, the oxygen transfer efficiency linearly upregulated in the range of 45-65 mmHg PaO 2 . In support, several reports of intestinal 16 or peritoneal 17 perfusion of oxygenated perfluorocarbon, albeit with incremental (-hourly) effectiveness, discussed that the most effective oxygenation was achieved at a pre-treatment PaO 2 level of around 40-60 mmHg.  iScience Article Interestingly, the correlation coefficient analysis indicates that the SvO 2 baseline is inversely associated with systemic oxygenation efficiency relative to pre-treatment arterial oxygenation status. This is in line with our mechanistic expectation since O 2 -PFD is initially exposed to venous circulation via the intestinal mucosa, thereby directly affecting oxygen transfer. Perfluorocarbon-dissolved O 2 is immediately available to deliver into hemoglobin by diffusion-based mechanisms. 18 With the intact intestinal epithelial cells, our experimental data favor that diffusion-based oxygen transfer into hemoglobin is a mechanism for the venous oxygen increase. Thus, ventilatory drive in mammalian enteral breathing likely involves recipient oxygenation status and should be considered when developing a clinical application for diseases of hypoxia.
Oxygen carriers for enteral ventilation include oxygen gas, oxygen microbubbles, and perfluorocarbons. Previous studies showed oxygen gas infusion requires mucosal damage to deliver sufficient oxygen 9 and, therefore, is unacceptable for humans. While oxygenated microbubble infusion is effective when given as a relatively high-dose bolus injection (i.e., 75-100 mL/kg: 3,750-5,000 mL for 50 kg), 10 the perfluorocarbon approach can deliver the benefit with modest, repeated, dosing (i.e., 20 mL/kg: 1000 mL for 50 kg). This injection volume has been within dosage readily approved for human enterography procedures in single-contrast technique. In this study, O 2 -PFD has been slowly infused by drop from a height within 50 cm to prevent a substantial increase in intrarectal pressure. However, the effects of O 2 -PFD on humans are unknown. Therefore, preclinical studies are planned to confirm the tolerability of intrarectal PFD in humans. Of the perfluorocarbon compounds, we elected to use perfluorodecalin (PFD), a perfluorocarbon already extensively used in animals 9 and human studies involving blood substitutes, liquid breathing, eye surgery, 19 and known for its extraordinary oxygen and carbon dioxide dissolving capacities. Consistent with the reported safety of PFD in humans, enteral O 2 -PFD exposure is safe and tolerable without significant complications such as colorectal perforation, bacterial translocation, or liver dysfunction.
Intrarectal bolus infusion of O 2 -PFD can be operated without a specialized respiratory care team even in resource-limited settings. Such affordable technique will provide a supplemental way to oxygenate until advanced medical care is available as a bridging therapy. Some potential scenarios include the prehospital transport, emergency, or the preparatory settings for the invasive respiratory support.

Limitations of the study
This study, however, has a few limitations. First, the observation was confined for up to 30 min; therefore, the duration of efficacy and cumulative risk is unclear. Second, PFD dose-dependent effect was not tested. Third, respirator settings remained constant throughout the procedures, precluding the assessment of the crosstalk between intra-and extrapulmonary respirations effects. Finally, since this study was carried out in pigs under general anesthesia and muscle relaxant, tolerability under an awake status requires future evaluation. Nevertheless, our present study, conducted using a combination of clinically relevant medical devices, including PFD and a rectal balloon tube, firmly supports the therapeutic potential of extrapulmonary ventilation. Further preclinical and clinical development of the enteral ventilation approach is warranted.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
The authors would like to thank Vivian Hwa for her critical reading of the manuscript and Kunihiko Takahashi for statistical advice. Illustrations were created using Biorender.com. We also appreciate Asuka Kodaka for generating graphical abstract. We appreciate Ryo Okabe, Shogo Nagata, Yasuyuki Kurihara, Koichi Fukumoto, Koichi Masuda, Asuka Kodaka, Masashi Takakura, Osamu Mitani, Yoshiro Yamamura, and the laboratory of IVTeC Co., Ltd. for technical assistance with the experiments, and experts at EVA Therapeutics, Inc., Mune Pharmaceutical Co., Ltd., and Maruishi Pharmaceutical Co., Ltd. for professional advice over the PFD manufacturing and preclinical study design. We also thank Mari Maezawa, Noriko Yokota, Michiko Mori, and all other Takebe Lab members for administrative and intellectual assistance. and female normal-sized pigs (41.6-43.2 kg, n = 2) were fasted for two days and given free access to water. A peroral intestinal irrigator (Niflecâ, EA Pharma Co., Ltd., Tokyo, Japan) was administered as a laxative two days before the experiment.

Hypoxia model
The hypoxic pig model was established by mixing oxygen and nitrogen using a gas flow meter in an anesthesia machine (FO-20A, ACOMA Medical Industry Co., Ltd., Tokyo, Japan). The inspiratory oxygen fraction (FiO 2 ) was adjusted to maintain the pulse oximeter (measuring SpO 2 ) at approximately 85%. Arterial blood gas analysis was initiated 10 min before experimental manipulation to confirm arterial oxygen saturation (SaO 2 ), partial pressure of oxygen in arterial blood (PaO 2 ), and partial pressure of carbon dioxide in arterial blood (PaCO 2 ) were stable.

Anesthesia, maintenance, and monitoring
A mixture of ketamine (10 mg/kg, intramuscular), xylazine (2 mg/kg, intramuscular), and isoflurane were used as anesthetics. After tracheal intubation, 2% isoflurane was used to maintain general anesthesia. After administering an intravenous bolus of rocuronium (25 mg), controlled mechanical ventilation was initiated under continuous intravenous rocuronium infusion (100 mg/h) (MSD Co. Ltd, Tokyo, Japan). To maintain the intravenous fluids, Ringer's lactate solution was administered at a dose of 5 mL/kg/h. In the case of pre-experimental hemodynamic instability, a 100 mL Ringer's lactate solution bolus was administered intravenously, and the maintenance fluid dose was increased to 10 mL/kg/h. If hemodynamic instability persisted after fluid loading, phenylephrine and/or epinephrine were continuously administered. Enteral ventilation protocol was initiated after the hemodynamics stabilized for more than 10 minutes.
The hypoxic pigs did not breathe spontaneously under continuous administration of high-dose muscle relaxants and were mechanically ventilated under controlled conditions. The ventilator (Spiritus; ACOMA Medical Industry Co., Ltd., Tokyo, Japan) was set at a tidal volume of 8-10 mL/kg and a respiratory rate of approximately 35 mmHg for end-tidal carbon dioxide. The peak end-expiratory pressure (PEEP) was set at 8-10 mmHg in the 5 head-up position to prevent deterioration of the lung condition, e.g., due to atelectasis. The ventilator settings remained constant throughout all experimental processes.
Catheters were placed in the right carotid and femoral arteries for repeated blood sampling and blood pressure monitoring, respectively. A Swan-Ganz catheter was placed in the pulmonary artery through the right jugular vein under fluoroscopy. Arterial pressure, pulmonary arterial pressure, SpO 2 , heart rate, blood temperature, and mixed venous oxygen saturation (SvO 2 ) were continuously monitored using a bedside patient monitor (Life Scope VS BSM-3592, Nihon Kohden Corporation, Tokyo, Japan). Cardiac output was measured intermittently using bolus thermodilution methods through a Swan-Ganz catheter before and after the intervention. iScience Article a rectal tube 30 min after O 2 -PFD infusion. Arterial blood gas analyses were performed every 2 min for 10 min after O 2 -PFD collection. This procedure was repeated three times. The experimental protocol is illustrated in Figure 1.

Enteral ventilation and blood gas analysis
In an additional experiment, O 2 -PFD (20 ml/kg) was administered into the distal intestine through a rectal balloon catheter (Create Medic Co., Ltd., Yokohama, Japan). Arterial, IVC, and portal vein blood samples were analyzed simultaneously every 3 minutes for up to 30 minutes after O 2 -PFD infusion. O 2 -PFD was collected by spontaneous discharge from the anus. Arterial blood gas analyses were performed every 3 min until 15 min after O 2 -PFD collection. These procedures were performed twice on each pig.
All blood gas analyses were performed using an ABL90 FLEX blood gas analyzer (Radiometer Medical ApS, Copenhagen, Denmark) equipped with a co-oximeter.

Outcome measurements
The effect of systemic oxygenation and ventilation were measured using arterial and pulmonary arterial blood gas analyses. Increases in oxygen saturation (SaO 2 , SvO 2 ) and arterial partial pressure (PaO 2 , PaCO 2 ) from baseline before administration of O 2 -PFD were evaluated. For each blood oxygenation parameter (PaO 2 , SaO 2 , SvO 2 ), the correlation between baseline status and oxygenation was assessed at each measurement interval after O 2 -PFD administration. The oxygen and carbon dioxide partial pressures of PFD were also measured before and after intestinal administration of O 2 -PFD.
To assess venous oxygenation levels, the portal vein and infrarenal level of IVC blood samples and pulmonary arterial blood samples were analyzed in an additional experiment using normal-sized pigs. Inspiratory oxygen and expiratory oxygen concentrations were measured by monitoring anesthetic and respiratory gases to evaluate oxygen discharge from expiration rather than inspiration (GF-119 Multi-Gas Unit, Nihon Kohden Corporation, Tokyo, Japan).
We used postmortem histopathology to assess whether the intestinal mucosa and spleen were damaged as a side effect of O 2 -PFD infusion. Vital signs, such as mean arterial pressure and pulse rate, were also monitored during the procedures.

QUANTIFICATION AND STATISTICAL ANALYSIS
The baseline characteristics of hypoxic pigs were compared using Student's t test, Mann-Whitney U test, or Fisher exact test. Systemic oxygenation and maximum ventilation effect by blood gas analysis before and after O 2 -PFD administration was evaluated using a paired t-test. The simultaneous increase in the oxygenation rate in arterial and mixed venous blood from baseline was compared using the Wilcoxon signed-rank test. The Pearson correlation coefficient evaluated the relationship between the pre-treatment condition and blood gas parameters after O 2 -PFD administration. The difference between inspiratory and exhaled oxygen concentrations was analyzed using a paired t-test. Categorical variables were expressed as numeric values (proportion), and continuous variables were presented as mean G standard deviation (SD) or median (interquartile range [IQR]). Delta (D) represents the difference in blood gas analysis values before and after O 2 -PFD administration. Statistical significance was set at p < 0.05. All statistical analyses were performed using Graph Pad Prism9 software and R software, version 4.1.2 (The R Foundation for Statistical Computing, Vienna, Austria).